Plants and insects have co-evolved in order to survive in their changing niches. Insects gradually adapt to take maximum nutritional benefit from the host while plants have evolved to defend themselves by up regulating the expression of defense related biochemicals (Bennett et al., 1994; Howe et al. 2008). In order to sustain on chemically varied dietary content, insects display molecular flexibilities resulting in their modified gut enzyme complement and metabolism (Koiwa et al., 1997; Kessler and Ian T. Baldwin, 2002; Srinivasan et al. 2006; Dawkar et al., 2011). Helicoverpa armigera (Lepidoptera: Noctuidae), which is an agronomically important insect pest, has been widely studied for its polyphagy and adaptability on various host plants (Patankar et al., 2001; Sarate et al., 2011).
Plant proteinase inhibitors (PIs) are ubiquitous in the plant kingdom and have been extensively studied as plant defense molecules which act by inhibiting hydrolytic enzymes from insect gut (Ryan, 1990; Damle et al., 2005). Among various serine proteinase inhibitor (PI) families, Pin-II/Pot-II family displays a remarkable structural and functional diversity at gene and protein level (Johnson et al. 1989; McManas et al. 1994; Duan et al. 1996; Barta et al., 2001; Kong and Rangnathan 2008). Wound, insect and stress induced up regulation of these PIs clearly link their function to plant defense. Several studies have been undertaken in the past few decades using transgenic systems or in vivo assays. These studies positively correlate the insect defensive advantage offered by Pin-II PI expression in plants (Green and Ryan, 1972; Agrawal, 1998; Zavala et al., 2004a and b). Recently Pin-II PIs from Nicotiana alata expressed as transgene in cotton and tested at the field level proved to enhance the productivity by 30% due to reduction in pest infestation (Dunse et al. 2010). In addition to the well-established defensive role, Pin-II PIs have been recently shown to have endogenous functions in plants which still remain to be fully elucidated (Sin and Chye, 2004; Wu et al., 2006; Johnson et al., 2007, Tamhane et al., 2009, Bezzi et al., 2010, Hartl et al., 2010).
Precursor proteins of Pin-II PIs consist of 1- to 8-inhibitory repeat domains (IRDs) connected by a protease sensitive linker, which upon cleavage releases IRD units. Each IRD is a peptide of around 50 amino acid with a molecular mass of ˜6 KDa. The amino acid sequences of inhibitory repeat domains show variations while the 8 cysteine residues and a single proline residue are almost conserved (Lee et al. 1999, Schirra et al. 2001, 2008 and 2010) throughout. Each IRD possesses a single active site either for trypsin or chymotrypsin inhibition based on the presence of lysine/arginine or leucine at the P1 position respectively. The Pin-II precursor and/or the IRDs are both capable of simultaneously inhibiting several or single protease molecule respectively (Lee et al., 1999, Tamhane et al., 2007, Mishra et al., 2010).
Structure of Pin-II PIs, either 2 domain precursor or individual IRD(s) have been studied (Nielsen et al. 1994, Barrette Ng et al. 2003, Schirra and Craik, 2005). IRD shows a disordered loop containing the reactive site, a triple stranded beta sheet at its base and is anchored by four conserved disulfide bonds (C4-C41, C7-C25, C8-C37 and C14-C50) (Scanlon et al., 1999, Schirra et al., 2001, Schirra et al., 2008). Among the four disulfide bonds, C8-C37 has been found to be very crucial for maintaining active conformation and hence inhibitory activity, whereas C4-C41 has important role in maintaining the flexibility of reactive loop (Schirra et al., 2010). Whereas selective loss of disulfide bond has evolutionary significance and leads to functional differentiation (Li et al, 2011).
In a standard mechanism of protease inhibition by Pin-II PIs, the convex shaped reactive loop of inhibitor (P1 side chain) is recognized by concave active site (S1 binding pocket) of enzyme in a substrate like manner and plays a major role in the energetics of recognition (Czapiñska & Otlewski, 1999, Otlewski et, al. 2001). Proteinase Inhibitor-proteinase interaction is further influenced by non-contact residues of the inhibitor by means of Van der Waals interaction and hydrogen (H) bonding. The structure of Pin-II IRDs or two domain PIs in complex with protease have been solved (Greenblatt et al. 1989, Barrette Ng et al. 2003b). The structure displays the molecular framework of the PI-protease interaction. Whereas structure of unbound Pin-II inhibitor gives information about conformational flexibility of reactive loop and its role in modulation of proteinase binding efficiency (Barrette Ng et al. 2003a). Thermodynamic analysis of protease-proteinase inhibitor interaction shows that it is entropy driven process (Otlewski et al., 2001). Different computational techniques like structure prediction, molecular dynamics and molecular docking studies have been used to study these interactions (Cui et al., 2005; Dunse et al., 2010).
Earlier studies have shown that Pin-II PIs from Capsicum annuum (CanPIs) and their recombinant proteins show anti-metabolic effects on the polyphagous and devastating insect pest H. armigera by inhibiting larval growth and development (Tamhane et al., 2005; 2007). CanPIs interact with the gut proteases of the H. armigera and are processed into their constituent IRDs (Mishra et al., 2010). 55 unique IRDs with amino acid variations in reactive loop and/or number of cysteine residues have been identified and characterized (Joshi et al., 2012; Mishra et al., 2012). Of these, the present inventors have selected three CanPI IRDs on the basis of amino acid sequence variation and deviation from the presence of 8 conserved cysteine residues.
The existing pest management strategies for controlling pests as described in the art are however putting very strong selective pressure on the insects thus leading to resistance. Therefore, there is a need to develop arena of effective and novel molecules, which can efficiently cause antibiosis of hazardous agricultural pest.